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The Journal of Neuroscience, September 15, 2002, 22(18):8028-8033
Distribution of Cystine/Glutamate Exchange Transporter, System
xc , in the Mouse Brain
Hideyo
Sato1,
Michiko
Tamba1,
Suzuka
Okuno1,
Kanako
Sato1,
Kazuko
Keino-Masu2, 3,
Masayuki
Masu2, and
Shiro
Bannai1
Departments of 1 Biochemistry and
2 Molecular Neurobiology, Institute of Basic Medical
Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan, and
3 Department of Physiology, National Defense Medical
College, Namiki, Saitama 359-8513, Japan
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ABSTRACT |
Mammalian cells express a transport system known as system
xc , which is an exchange agency
specific for anionic forms of cystine and glutamate. System
xc activity is important to maintain
both intracellular glutathione levels and the redox balance between
cystine and cysteine in the extracellular milieu. We have shown that
the cloned cDNAs encoding the transporter for system
xc consist of two components, xCT and
the heavy chain of 4F2 antigen. In the present study, we have
investigated the mRNA distribution for these components in the mouse
brain by in situ hybridization. The xCT mRNA was
expressed in the area postrema, subfornical organ, habenular nucleus,
hypothalamic area, and ependymal cells of the lateral wall of the third
ventricle in the adult mouse brain. A strong signal was also detected
in the meninges in both adult and fetal mouse brains. The mRNA
expression of the heavy chain of 4F2 antigen was detected in a more
broad area, including all of the regions in which xCT mRNA was
detected. These data are compatible with our biochemical evidence that
xCT functions in combination with the heavy chain of 4F2 antigen to
elicit system xc activity. The
expression of system xc in meninges
and some circumventricular organs may suggest that this transporter
contributes to the maintenance of the redox state (i.e.,
cysteine/cystine ratio) in the CSF.
Key words:
system xc; cystine; glutamate; glutathione; amino acid transport; CSF; circumventricular
organ
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INTRODUCTION |
Transport of amino acids across
plasma membrane is mediated by several transport systems in mammalian
cells (Christensen, 1990 ). We have described an
Na+-independent, anionic amino acid
transport system that is highly specific for cystine and glutamate in
cultured human fibroblasts and mouse peritoneal macrophages (Bannai and
Kitamura, 1980; Watanabe and Bannai, 1987). This system, designated as
system xc, transports
an anionic form of cystine in exchange for glutamate (Bannai, 1986).
The activity of system
xc is drastically
induced by electrophilic agents and oxygen in various cultured cells
(Bannai, 1984a; Bannai et al., 1986, 1989; Miura et al., 1992). The
cells expressing system
xc take up cystine in
the medium and reduce it to cysteine (sulfhydryl form), which is in
turn used for the synthesis of glutathione and proteins. A part of
cysteine is released back into the medium via neutral amino acid
transport systems; then cysteine is rapidly oxidized to cystine by
oxygen in the medium (Bannai et al., 1989). Thus, the activity of
system xc contributes
to driving the cystine/cysteine cycle and to maintaining the balance
between cystine and cysteine in the culture medium. In these cultured
cells, the activity of system
xc is also demonstrated
to be essential for maintaining the intracellular glutathione levels
(Bannai and Tateishi, 1986). Because glutathione is one of the cellular
antioxidant systems and plays a central role in eliminating oxidative
stress, system xc is
categorized as the antioxidant defense system, at least in cultured
cells. However, it is unknown whether system
xc functions as an
antioxidant defense system in vivo.
We have recently isolated cDNAs for this transporter and found that it
is composed of two protein components, the surface antigen of 4F2 heavy
chain (4F2hc) and a novel protein named xCT (Sato et al., 1999). xCT
has 12 putative transmembrane domains, whereas 4F2hc is predicted to
have a single transmembrane domain. It has been shown that in some
amino acid transporters, such as systems L and
y+L, 4F2hc brings the counterpart
light-chain proteins to the surface of the plasma membrane
(Mastroberardino et al., 1998; Torrents et al., 1998). We have shown
that the combined expression of xCT and 4F2hc is indispensable for
eliciting xc activity
in Xenopus oocytes (Sato et al., 1999). Thus, it is likely
that 4F2hc functions similarly in system
xc. In Northern blot
analysis, the mRNA for xCT was detected in the mouse brain, but not in
the heart, liver, lung, or kidney (Sato et al., 1999). The activity of
system xc has been
found in primary cultures of rat cortical neurons (Murphy et al., 1990;
Ishige et al., 2001), isolated brain cells from fetal rats (Sagara et
al., 1993), and human glioma cells (Ye et al., 1999). In the present
study, we have investigated the distribution of xCT and 4F2hc mRNAs in
the mouse brain by in situ hybridization. The data indicate
that system xc is
robustly expressed in some specific brain regions facing the cerebral
ventricles and in meninges, suggesting that this transporter is
involved in maintaining the redox state of the CSF in the brain.
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MATERIALS AND METHODS |
Mouse xCT cDNA digested with PstI (34-369 bp), mouse
xCT cDNA digested with BamHI and HindIII
(886-1843 bp), and mouse 4F2hc cDNA digested with PstI and
EcoRI (948-1793 bp) were subcloned into pBluescript. Each
cRNA probe was digoxigenin (DIG)-labeled by transcription from the
linearized plasmid using RNA-labeling mix (Roche Diagnostics GmbH,
Mannheim, Germany) and T3/T7 RNA polymerase (Stratagene, La
Jolla, CA). The in situ hybridization was performed as
described previously (Ohto et al., 2002). Female C57BL/6Cr mice
weighing 18-21 gm were deeply anesthetized with sodium pentobarbital
(20 mg/kg, i.p.), perfused, and fixed with 4% paraformaldehyde in PBS,
pH 7.4. The brains were excised and postfixed in the same fixative
overnight. Then the brains were incubated in 30% sucrose in PBS
overnight and embedded with optimal cutting temperature (OCT)
compound (Sakura Finetechnical Co., Ltd., Tokyo, Japan). Sections (10 µm) were cut in a cryostat and picked up on
Matsunami-aminosilane-coated microscope slides (Matsunami, Osaka, Japan). To remove the OCT compound, the slides were placed in
PBS containing 0.1% Tween 20 (PBT) at room temperature twice for 5 min
and then incubated in PBT containing 1 gm/ml proteinase K at 37°C for
5 min. The slides were rinsed in PBT three times, fixed with 4%
paraformaldehyde, rinsed in PBT three times, and incubated with
hybridization buffer (50% formamide, 5× SSC, pH 4.5, 1% SDS, 50 µg/ml heparin, and 50 µg/ml yeast RNA). After the addition of probe
(1000 ng/ml), slides were hybridized at 65°C overnight. Slides were
rinsed in 50% formamide, 5× SSC, pH 4.5, 1% SDS at 65°C for 30 min; in 50% formamide, 2× SSC at 65°C three times for 30 min each;
and with 25 mM Tris-HCl, pH 7.5, 0.8% NaCl,
0.02% KCl, 0.1% Tween 20 (TBST) at room temperature three times for 5 min each. Slides were submerged in the blocking buffer [0.5% blocking
reagent (Roche) in TBST] at room temperature for 1 hr and then
incubated in sheep anti-DIG antibody conjugated to alkaline phosphatase
in the blocking buffer at 4°C overnight. Slides were rinsed with TBST
containing 2 mM levamisole at room temperature
three times for 20 min each. After a 5 min rinse in 0.1 M NaCl, 50 mM Tris, pH 9.5, 50 mM MgCl2, 0.1% Tween
20, and 2 mM levamisole, slides were developed in
the dark in BM purple alkaline phosphatase substrate solution
(Roche) containing 2 mM levamisole for 2 d.
The development was stopped by rinsing several times with 10 mM Tris and 1 mM EDTA.
In Northern blot analysis, total RNA was isolated from several mouse
brain regions, dissected under a microscope, and electrophoresed on a
1% agarose gel in the presence of 2.2 M formaldehyde. Then the RNA was transferred onto positively charged nylon membranes (Roche)
and hybridized with the DIG-labeled RNA probes in DIG Easy Hyb (Roche)
for 16 hr at 68°C. The membrane was washed twice for 5 min at room
temperature with 2× SSC and 0.1% SDS and then washed twice for 15 min
at 68°C with 0.1× SSC and 0.1% SDS. In Northern blot analysis using
32P-labeled DNA probes, the RNA was
transferred onto Hybond N+ membrane
(Amersham Biosciences, Arlington Heights, IL) and hybridized in a
solution containing 50% formamide for 16 hr at 42°C. The membrane
was washed twice for 15 min at room temperature with 1× SSC and 0.1%
SDS and then washed twice for 15 min at 68°C with 0.25 or 0.1× SSC
and 0.1% SDS. The DNA probes were labeled using [ -32P]dCTP and the Rediprime II
random prime labeling system (Amersham Biosciences). The templates of
the 32P-labeled DNA probes were
PCR-amplified fragments between 324 and 1461 bp of mouse xCT cDNA and
221 and 1478 bp of mouse 4F2hc cDNA, respectively.
In reverse transcription PCR (RT-PCR), the first-strand
cDNA was synthesized from 1 µg of the total RNA isolated from several mouse brain regions dissected under a microscope using
oligo-dT12-18 as a primer. PCR amplification of
the cDNA was performed using the primer set 5'-CTCGTGACAGCTGTGGGCAT-3'
and 5'-GGCACTAGACTCAAGAACTGTG-3', corresponding to the nucleotide
sequences of mouse xCT (GenBank/European Molecular Biology
Laboratory/DNA Databank of Japan accession no. AB022345).
The experimental procedures involving animals were approved by the
University of Tsukuba Animal Care and Use Committee and were done in
accordance with its guidelines.
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RESULTS |
Expression of xCT and 4F2hc mRNAs in the mouse brain was
investigated by nonisotopic in situ hybridization using
DIG-labeled riboprobes. The antisense probes for both xCT (a
BamHI-HindIII fragment of 886-1843 bp) and
4F2hc (a PstI-EcoRI fragment of 948-1793 bp)
detected their mRNA expression in some brain regions, whereas the sense
probes used as negative controls gave rise to no signal (Figs.
1, 2). As
shown in Figure 1A-D, both xCT and 4F2hc mRNAs were
robustly expressed in approximately one-half of the cells in the area
postrema (AP) and in one of the circumventricular organs (CVOs); weaker
expression of these mRNAs was observed in the nucleus of the solitary
tract. Positive signals for both xCT and 4F2hc were also detected in
the subfornical organ (SFO), another CVO (Fig. 2). In addition, the
4F2hc signal was detected in the choroid plexus, cerebellum, brainstem,
and thalamus, whereas the signal for xCT was hardly detected in these
regions (Figs. 1, 2). Besides CVOs, strong expression of xCT mRNA
was observed in the ependymal cells located in the single
layer of the ventricular wall adjacent to the ventromedial hypothalamic
area (Fig. 3A,B). Expression
of the 4F2hc mRNA was also detected in the ependymal cells in the same
region (Fig. 3D,E). In the hypothalamic area, positive
signals for both xCT and 4F2hc were observed in the scattered cells
(Fig. 3C,F), although it remains undetermined which
cell types are expressing these mRNAs. Strong expression of xCT was detected in medial habenular nuclei and paraventricular thalamic nuclei
facing the dorsal third ventricle (Fig.
4A,C), whereas the
4F2hc signal was more broadly detected in these regions (Fig. 4B,D).
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Figure 1.
Expression of xCT and 4F2hc mRNAs in the AP of the
adult mouse brain detected by nonisotopic in situ
hybridization. Adjacent sagittal sections were hybridized with
DIG-labeled antisense riboprobes for xCT (A, C) and
4F2hc (B, D), respectively. C and
D are magnifications of the boxed regions
in A and B, respectively. No signal was
detected with the sense probes for xCT and 4F2hc, respectively
(E, F). Cb, Cerebellum;
CP, choroid plexus; Sol, nucleus of the
solitary tract. Scale bars: A, B, E, F, 200 µm;
C, D, 50 µm.
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Figure 2.
Expression of xCT and 4F2hc mRNAs in the SFO of
the adult mouse brain detected by nonisotopic in situ
hybridization. Adjacent coronal sections were hybridized with
DIG-labeled antisense riboprobes for xCT (A, C) and
4F2hc (B, D), respectively. C and
D are magnifications of the boxed regions
in A and B, respectively.
CC, Corpus callosum; CP, choroid plexus.
Scale bars: A, B, 200 µm; C, D, 50 µm.
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Figure 3.
Expression of xCT and 4F2hc mRNAs in the ependymal
cells of the lateral wall of the third ventricle
(3V) and in the hypothalamic area of the adult
mouse brain detected by nonisotopic in situ
hybridization. Adjacent coronal sections were hybridized with
DIG-labeled antisense riboprobes for xCT (A-C)
and 4F2hc (D-F). B, C and
E, F are magnifications of the boxed
regions with solid lines and broken
lines in A and D, respectively.
Scale bars: A, D, 200 µm; B, C, E, F,
50 µm.
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Figure 4.
Expression of xCT and 4F2hc mRNAs in the medial
habenular nucleus (MHb) of the adult mouse brain
detected by nonisotopic in situ hybridization. Adjacent
coronal sections were hybridized with DIG-labeled antisense riboprobes
for xCT (A, C) and 4F2hc (B, D).
C and D are magnifications of the
boxed regions in A and B,
respectively. CP, Choroid plexus; DG,
hippocampal dentate gyrus; PV, paraventricular thalamic
nucleus. Scale bars: A, B, 200 µm; C,
D, 50 µm.
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Outside the brain parenchyma, the meninges showed high levels of
expression of xCT and 4F2hc mRNAs (Fig.
5). Figure 5A-D shows representative examples of xCT and 4F2hc mRNA expression in
the meninges lining the surface of the posterior colliculus and
cerebellum. Judging from the distribution of the signal, xCT and 4F2hc
expression appears to be present in the arachnoid and pia matters (Fig.
5C,D). In other areas, such as the one ventral to the
hypothalamus, the signals for both xCT and 4F2hc were strongly detected
in the meninges but not in the cells of the large blood vessels (Fig.
5E,F). To investigate the ontogeny of xCT mRNA
expression, we performed in situ hybridization of the
embryonic mouse brain. The signal for xCT mRNA was detected primarily
in the meninges surrounding the brain of the embryonic day 15 mouse,
with especially high expression in the olfactory bulb (Fig.
6A,B) and in the
subarachnoid space of the cisterna magna (Fig. 6C,D). The
signal of 4F2hc mRNA was detected throughout the brain regions,
including the meninges (data not shown).
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Figure 5.
Expression of xCT and 4F2hc mRNAs in the meninges
of the adult mouse brain detected by nonisotopic in situ
hybridization. Adjacent sagittal sections were hybridized with
DIG-labeled antisense riboprobes for xCT (A, C, E) and
4F2hc (B, D, F). C and
D are magnifications of the boxed regions
in A and B, respectively.
E and F show the expression in the
meninges underneath the hypothalamus. Cb, Cerebellum;
Cx, cerebral cortex; PC, posterior
colliculus. Scale bars: A, B, 200 µm;
C-F, 50 µm.
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Figure 6.
Expression of xCT and 4F2hc mRNAs in the mouse
embryos detected by nonisotopic in situ hybridization.
Sagittal sections of the embryonic day 15 embryos were hybridized with
the DIG-labeled antisense xCT riboprobe (A-D).
B and D are magnifications of the
boxed regions of the olfactory lobe region in
A and in the cisterna magna in C.
Br, Brain; OB, olfactory bulb;
T, tongue. Scale bars: A, C, 800 µm;
B, 200 µm; D, 100 µm.
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To test the specificity of the data obtained in the above experiments,
we have adopted two different approaches. First, we performed in
situ hybridization using another set of antisense and sense probes
for xCT (a PstI fragment of 34-369 bp), which do not
overlap with those used in Figures 1-6. The antisense probe for xCT
detected the signal in the AP, whereas the sense probe did not give any
signal (Fig. 7). This result is identical
to the one obtained using the original probes. Next, we performed Northern blot analysis using the total RNA isolated from mouse brains.
To this end, we carefully dissected five small regions from the adult
mouse brain under a microscope, and total RNA was isolated. These RNAs
were hybridized with the DIG-labeled RNA probes, which were used in the
in situ hybridization experiments in this study, and also
with the 32P-labeled DNA probe used
previously (Sato et al., 1999). Both probes detected a strong signal in
the meninges (Fig. 8A),
which is in accordance with the high expression of xCT in the meninges in the in situ hybridization experiments. As shown
previously, mRNA for xCT is expressed as the multiple bands, ~12,
3.5, and 2.5 kb, which probably represent alternative splicing,
alternative polyadenylation sites, or a combination of both. The
prominent 12 kb band has been suggested to represent mRNA with
long 3'-untranslated regions (Sato et al., 1999, 2000). The size of the
band is the same as the one observed in the activated macrophage used
as a positive control. Weak expression was detected in the hypothalamus and the cerebral cortex, and very faint signals were observed in the
cerebellum and choroid plexus (Fig. 8A). The 4F2hc
signal was detected in all of the areas examined, which is also
compatible with the data obtained in the in situ
hybridization experiments. To confirm the results of the Northern blot
analysis, we performed RT-PCR using the total RNA from the five small
regions from the adult mouse brain (Fig. 8B). A
strong signal was detected in the meninges; weak expression was
detected in the hypothalamus and the cerebral cortex. Very faint
signals were observed in the cerebellum and choroid plexus. These
results are consistent with those of the Northern blot analysis. The
strong signal of the macrophage sample is thought to be attributable to
the relatively large amount of smaller mRNAs for xCT (3.5 and 2.5 kb)
in the macrophages (Sato et al., 1999).
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Figure 7.
Expression of xCT mRNA in the AP of the adult
mouse brain detected by nonisotopic in situ
hybridization. Adjacent sagittal sections were hybridized with
DIG-labeled antisense (A) and sense
(C) riboprobes for xCT. The probes used in this
experiment do not overlap with those used in Figures 1-6.
B is the magnification of the boxed
region in A. Cb, Cerebellum;
CP, choroid plexus; Sol, nucleus of the
solitary tract. Scale bars: A, C, 200 µm;
B, 50 µm.
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Figure 8.
Expression of xCT and 4F2hc mRNAs in various areas
of the adult mouse brain. A, Northern blot analysis of
xCT and 4F2hc. Total RNA (1.5 µg) isolated from the choroid
plexus, hypothalamus, meninges, cortex, cerebellum, whole brain, and
the mouse peritoneal macrophage cultured for 8 hr with 1 ng/ml
bacterial lipopolysaccharide was loaded per lane. Hybridization was
performed with DIG-labeled RNA probes or 32P-labeled DNA
probes for xCT, 4F2hc, and  -actin. The DIG-labeled RNA probes for
xCT and 4F2hc are the same as those used in Figures 1-6.
B, RT-PCR of total RNA from the choroid plexus,
hypothalamus, meninges, cortex, cerebellum, whole brain, and the mouse
peritoneal macrophage cultured for 8 hr with 1 ng/ml bacterial
lipopolysaccharide. The figure shows the ethidium bromide-stained
agarose electrophoresis of the PCR products using the primer sets
corresponding to the sequence of xCT (top) and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH;
bottom).
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DISCUSSION |
In the present study, we have demonstrated that xCT and 4F2hc
mRNAs are especially abundant in several regions facing the CSF. Thus,
it is likely that system
xc in these regions
contributes to the clearance of cystine from the CSF. Cysteine and
cystine, or glutathione and glutathione disulfide, are the critical
components as cellular redox buffers in metabolism and homeostasis
(Deneke, 2000). In the blood plasma, cysteine and cystine are the main
redox components, and the ratio of cysteine/cystine is kept constant
(Kleinman and Richie, 2000). The total concentration of free cysteine
and cystine in the plasma is ~120 µM (as half-cystine),
and 10-20% of the total is present as the reduced form (Bannai,
1984b; Andersson et al., 1995), although some disturbance was observed
in patients with end-stage renal failure (Wlodek et al., 2001). In
contrast, the data available for the concentration of cysteine and
cystine in the CSF are limited. Only total cystine (cystine plus
cysteine) was determined and was reported to be 0.25-1.3
µM in human CSF (Lakke and Teelken, 1976; Araki et al.,
1988). Recently, using the thiol-specific fluorogenic reagent, the
concentration of cysteine in human CSF was determined to be ~2.5
µM (Castagna et al., 1995). In rats, the concentration of
cysteine in CSF has been demonstrated to be ~4 µM
(Anderson et al., 1989). Wang and Cynader (2000) have shown that the
concentration of cysteine in the CSF of rats ranges from 0.68 to 1.75 µM (average, 1.12 µM), whereas cystine is
not detected. Together, the ratio of cysteine to cystine in CSF is very
high compared with that in plasma. Data for the auto-oxidation rate of
cysteine in the CSF are not available; the stability of cysteine in the
CSF remains to be investigated. Data for the glutathione level in CSF
are also limited; the reported values are very variable. Concentrations
of glutathione in rat and human CSF are ~5-6 µM (Anderson et al., 1989; Wang and Cynader, 2000) and 0.2-0.3
µM (Castagna et al., 1995; Konings et al., 1999),
respectively. In the brain, reactive oxygen species are produced at a
higher rate because of the high consumption of oxygen. Recent studies
have suggested that the reactive oxygen species are involved in the pathogenesis of various neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (Bains and Shaw, 1997). Because cerebral ventricles, meninges, and the extracellular space in the parenchyma are filled with
CSF, the antioxidant potential of CSF seems to be important. The
cysteine/cystine balance may serve as a major redox buffer in the CSF.
Clearance of cystine via system
xc in the cells facing
the CSF and release of cysteine from the cells via neutral amino acid
transporter(s) may contribute to maintaining the reduced state in the
CSF against the oxidative stress. Figure
9 shows the hypothetical model for system
xc in maintaining the
redox state of the CSF; a similar model has long been established in
the cell-culture system (Bannai et al., 1989). System
xc is an exchange
agency, and the anionic form of cystine is transported in exchange for
glutamate. The exchange is obligatory, with a molar ratio of 1:1. One
of the glutamate transporters, GLAST (also called EAAT 1), is
expressed in the AP, SFO, and meninges (Berger and Hediger, 2000). This
transporter may contribute to the reabsorption of glutamate effluxed
from the cells in exchange for cystine via system
xc.
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Figure 9.
Schematic model for the cystine/cysteine cycle in
the subarachnoid space. Cystine (Cyss) in the CSF is
taken up by system xc and reduced to
cysteine (CySH), which is primarily used for
glutathione (GSH) synthesis; alternatively, it is
taken out of the cell by neutral amino acid transporters. Although
cysteine in the CSF is rapidly oxidized to cystine, it is cleared from
the CSF in the subarachnoid space by the action of system
xc. Glu,
Glutamate.
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CVOs are highly vascularized structures; they possess unusual vascular
arrangements, with many capillary loops reaching near the ventricular
surface (Oldfield and McKinley, 1995). These capillaries have
fenestrated endothelial cells, resulting in local disruption of the
blood-brain barrier. AP and SFO contain receptors or binding sites for
many peptides, including angiotensin, and participate in the general
regulation of fluid balance and blood pressure (Fitzsimons, 1998). In
addition, these regions contain neuronal perikarya and are referred to
as the sensory CVOs (Johnson and Gross, 1993). They have extensive
afferent and efferent neural networks between the CVOs and other brain
structures. There can be bidirectional movement of polar molecules
between the blood, the CSF, and the brain parenchyma. Thus, these
regions may be allowed to detect small changes in circulating hormones,
peptides, and neuroactive substances and to respond hormonally and
neurally to these signals. System
xc expressed in the SFO
and the AP may function as the sensor for the redox balance between
cysteine and cystine in the blood and/or the CSF.
4F2hc functions as the common component not only for system
xc but also for systems
L and y+L to elicit their activities
(Verrey et al., 2000). In our study, 4F2hc mRNA was detected in all of
the areas in the mouse brain where xCT mRNA was expressed, including
the AP, SFO, lateral wall of the third ventricle, medial habenular
nuclei, and meninges. 4F2hc mRNA was also detected in areas such as the
hippocampus, choroid plexus, cortex, and cerebellum. Thus, 4F2hc may be
coupled with other partners to elicit different transporter activities. Among the transporters that require 4F2hc for the expression of their
transporter activity, L-type amino acid transporter-2 (LAT-2) mediates the transport for neutral amino acids, including cysteine; its
mRNA is detected in the brain (Segawa et al., 1999). It is likely that
LAT-2 mRNA is expressed in the areas where 4F2hc mRNA is detected and
that the activity of system L is expressed in those areas. Although the
significant signal of xCT mRNA was not detected in the cortex or
cerebellum by our nonisotopic in situ hybridization, the
results do not exclude the possibility that a low level of xCT mRNA is
expressed in these areas. Indeed, very weak signals were detected in
these brain areas by Northern blot analysis and by RT-PCR, and the
activity of system xc
has been demonstrated in cortical neurons (Murphy et al., 1990; Ishige
et al., 2001) and in the cerebellum (Wyatt et al., 1996; Warr et al.,
1999).
Alzheimer's disease is associated with the intraparenchymal growth of
plaque-like amyloid deposits (Sisodia and Price, 1995), which
are composed of the amyloid peptide (A). A is derived by
proteolytic cleavage of the amyloid protein precursor (APP), which
contains a cysteine-rich region. Both A and APP are found in the CSF
(Seubert et al., 1992). Huang et al. (1999) have demonstrated that A
is capable of generating hydrogen peroxide by metal ion reduction. The
reduced state in the CSF might be important for quenching hydrogen
peroxide produced by A. The human xCT is localized at chromosome 4q28-31 (Sato et al., 2000). In this locus, the gene for
glycophorin A is located, and a link with Alzheimer's disease has been
suspected (Race, 1959), although close linkage of this gene with
Alzheimer's disease has been excluded (Spence et al., 1986). In the
cultured cells, the enhanced activity of system
xc increases the
intracellular glutathione level. If system
xc functions similarly
in vivo, several regions expressing system xc may contain higher
levels of glutathione, which contributes to the decrease in the
oxidative stress produced in the CSF and parenchyma. The involvement of
system xc in
neurodegenerative diseases deserves additional exploration.
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FOOTNOTES |
Received April 8, 2002; revised July 2, 2002; accepted July 9, 2002.
This work was supported by a Grant-in-Aid for scientific research from
the Ministry of Education, Culture, Sports, Science, and Technology of
Japan. We thank Dr. S. Hisano for helpful discussions.
Correspondence should be addressed to Dr. Hideyo Sato, Department of
Biochemistry, Institute of Basic Medical Sciences, University of
Tsukuba, Tsukuba, Ibaraki 305-8575, Japan. E-mail:
hideyo-s{at}md.tsukuba.ac.jp.
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REFERENCES |
-
Anderson ME,
Underwood M,
Bridges RJ,
Meister
(1989)
Glutathione metabolism at the blood-cerebrospinal fluid barrier.
FASEB J
3:2527-2531[Abstract].
-
Andersson A,
Lindgren A,
Hultberg B
(1995)
Effect of thiol oxidation and thiol export from erythrocytes on determination of redox status of homocysteine and other thiols in plasma from healthy subjects and patients with cerebral infarction.
Clin Chem
41:361-366[Abstract/Free Full Text].
-
Araki K,
Harada M,
Ueda Y,
Takino T,
Kuriyama K
(1988)
Alteration of amino acid content of cerebrospinal fluid from patients with epilepsy.
Acta Neurol Scand
78:473-479[Web of Science][Medline].
-
Bains JS,
Shaw CA
(1997)
Neurodegenerative disorders in humans: the role of glutathione in oxidative stress-mediated neuronal death.
Brain Res Brain Res Rev
25:335-358[Medline].
-
Bannai S
(1984a)
Induction of cystine and glutamate transport activity in human fibroblasts by diethyl maleate and other electrophilic agents.
J Biol Chem
259:435-2440[Abstract/Free Full Text].
-
Bannai S
(1984b)
Transport of cystine and cysteine in mammalian cells.
Biochim Biophys Acta
779:289-306[Medline].
-
Bannai S
(1986)
Exchange of cystine and glutamate across plasma membrane of human fibroblasts.
J Biol Chem
261:2256-2263[Abstract/Free Full Text].
-
Bannai S,
Kitamura E
(1980)
Transport interaction of L-cystine and L-glutamate in human diploid fibroblasts in culture.
J Biol Chem
255:2372-2376[Free Full Text].
-
Bannai S,
Tateishi N
(1986)
Role of membrane transport in metabolism and function of glutathione in mammals.
J Membr Biol
89:1-8[Web of Science][Medline].
-
Bannai S,
Takada A,
Kasuga H,
Tateishi N
(1986)
Induction of cystine transport activity in isolated rat hepatocytes by sulfobromophthalein and other electrophilic agents.
Hepatology
6:1361-1368[Web of Science][Medline].
-
Bannai S,
Sato H,
Ishii T,
Sugita Y
(1989)
Induction of cystine transport activity in human fibroblasts by oxygen.
J Biol Chem
264:18480-18484[Abstract/Free Full Text].
-
Berger UV,
Hediger MA
(2000)
Distribution of the glutamate transporters GLAST and GLT-1 in rat circumventricular organs, meninges, and dorsal root ganglia.
J Comp Neurol
421:385-399[Medline].
-
Castagna A,
Le Grazie C,
Accordini A,
Giulidori P,
Cavalli G,
Bottiglieri T,
Lazzarin A
(1995)
Cerebrospinal fluid S-adenosylmethionine (SAMe) and glutathione concentrations in HIV infection: effect of parenteral treatment with SAMe.
Neurology
45:1678-1683[Abstract/Free Full Text].
-
Christensen HN
(1990)
Role of amino acid transport and countertransport in nutrition and metabolism.
Physiol Rev
70:43-77[Free Full Text].
-
Deneke SM
(2000)
Thiol-based antioxidants.
In: Current topics in cellular regulation, Vol 36 (Stadtman ER,
Chock PB,
eds), pp 151-180. San Diego: Academic.
-
Fitzsimons JT
(1998)
Angiotensin, thirst, and sodium appetite.
Physiol Rev
78:583-686[Abstract/Free Full Text].
-
Huang X,
Atwood CS,
Hartshorn MA,
Multhaup G,
Goldstein LE,
Scarpa RC,
Cuajungco MP,
Gray DN,
Lim J,
Moir RD,
Tanzi RE,
Bush AI
(1999)
The A peptide of Alzheimer's disease directly produces hydrogen peroxide through metal ion reduction.
Biochemistry
38:7609-7616[Medline].
-
Ishige K,
Chen Q,
Sagara Y,
Schubert D
(2001)
The activation of dopamine d4 receptors inhibits oxidative stress-induced nerve cell death.
J Neurosci
21:6069-6076[Abstract/Free Full Text].
-
Johnson AK,
Gross PM
(1993)
Sensory circumventricular organs and brain homeostatic pathways.
FASEB J
7:678-686[Abstract].
-
Kleinman WA,
Richie Jr JP
(2000)
Status of glutathione and other thiols and disulfides in human plasma.
Biochem Pharmacol
60:19-29[Web of Science][Medline].
-
Konings CH,
Kuiper MA,
Teerlink T,
Mulder C,
Scheltens PH,
Wolters ECH
(1999)
Normal cerebrospinal fluid glutathione concentrations in Parkinson's disease, Alzheimer's disease, and multiple system atrophy.
J Neurol Sci
168:112-115[Web of Science][Medline].
-
Lakke JPWF,
Teelken AW
(1976)
Amino acid abnormalities in cerebrospinal fluid of patients with parkinsonism and extrapyramidal disorders.
Neurology
26:489-493[Abstract/Free Full Text].
-
Mastroberardino L,
Spindler B,
Pfeiffer R,
Skelly PJ,
Loffing J,
Shoemaker CB,
Verrey F
(1998)
Amino-acid transport by heterodimers of 4F2hc/CD98 and members of a permease family.
Nature
395:288-291[Medline].
-
Miura K,
Ishii T,
Sugita Y,
Bannai S
(1992)
Cystine uptake and glutathione level in endothelial cells exposed to oxidative stress.
Am J Physiol
262:C50-C58[Abstract/Free Full Text].
-
Murphy TH,
Schnaar RL,
Coyle JT
(1990)
Immature cortical neurons are uniquely sensitive to glutamate toxicity by inhibition of cystine uptake.
FASEB J
4:1624-1633[Abstract].
-
Ohto T,
Uchida H,
Yamazaki H,
Keino-Masu K,
Tatsui A,
Masu M
(2002)
Identification of a novel nonlysosomal sulphatase expressed in the floor plate, choroids plexus, and cartilage.
Genes Cells
7:173-186[Abstract].
-
Oldfield BJ,
McKinley MJ
(1995)
Circumventricular organs.
In: The rat nervous system (Paxinos G,
ed), pp 391-403. San Diego: Academic.
-
Race RR
(1959)
Familial Alzheimer's disease: note on the linkage data.
Ann Hum Genet
23:310.
-
Sagara J,
Miura K,
Bannai S
(1993)
Cystine uptake and glutathione level in fetal brain cells in primary culture and in suspension.
J Neurochem
61:1667-1671[Web of Science][Medline].
-
Sato H,
Tamba M,
Ishii T,
Bannai S
(1999)
Cloning and expression of a plasma membrane cystine/glutamate exchange transporter composed of two distinct proteins.
J Biol Chem
274:11455-11458[Abstract/Free Full Text].
-
Sato H,
Tamba M,
Kuriyama-Matsumura K,
Okuno S,
Bannai S
(2000)
Molecular cloning and expression of human xCT, the light chain of amino acid transport system xc.
Antioxid Redox Signal
2:665-671[Medline].
-
Segawa H,
Fukasawa Y,
Miyamoto K,
Takeda E,
Endou H,
Kanai Y
(1999)
Identification and functional characterization of a Na+-independent neutral amino acid transporter with broad substrate selectivity.
J Biol Chem
274:19745-19751[Abstract/Free Full Text].
-
Seubert P,
Vigo-Pelfrey C,
Esch F,
Lee M,
Dovey H,
Davis D,
Sinha S,
Scholossmacher M,
Whaley J,
Swindlehurst C,
McCormark R,
Wolfert R,
Selkoe D,
Lieberburg I,
Schenk DB
(1992)
Isolation and quantification of soluble Alzheimer's -peptide from biological fluids.
Nature
359:325-327[Medline].
-
Sisodia SS,
Price DL
(1995)
Role of the -amyloid protein in Alzheimer's disease.
FASEB J
9:366-370[Abstract/Free Full Text].
-
Spence MA,
Heyman A,
Marazita ML,
Sparkes RS,
Weinberg T
(1986)
Genetic linkage studies in Alzheimer's disease.
Neurology
36:581-584[Abstract].
-
Torrents D,
Estévez R,
Pineda M,
Fernández E,
Lloberas J,
Shi YB,
Zorzano A,
Palacín M
(1998)
Identification and characterization of a membrane protein (y+L amino acid transporter-1) that associates with 4F2hc to encode the amino acid transport activity y+L: a candidate gene for lysinuric protein intolerance.
J Biol Chem
273:32437-32445[Abstract/Free Full Text].
-
Verrey F,
Meier C,
Rossier G,
Kühn LC
(2000)
Glycoprotein-associated amino acid exchangers: broadening the range of transport specificity.
Pflügers Arch
440:503-512[Web of Science][Medline].
-
Wang XF,
Cynader MS
(2000)
Astrocytes provide cysteine to neurons by releasing glutathione.
J Neurochem
74:1434-1442[Web of Science][Medline].
-
Warr O,
Takahashi M,
Attwell D
(1999)
Modulation of extracellular glutathione in rat brain slices by cystine-glutamate exchange.
J Physiol (Lond)
514:783-793[Abstract/Free Full Text].
-
Watanabe H,
Bannai S
(1987)
Induction of cystine transport activity in mouse peritoneal macrophages.
J Exp Med
165:628-640[Abstract/Free Full Text].
-
Wlodek PJ,
Iciek MB,
Milkowski A,
Smolenski OB
(2001)
Various forms of plasma cysteine and its metabolites in patients undergoing hemodialysis.
Clin Chim Acta
304:9-18[Medline].
-
Wyatt I,
Gyte A,
Simpson MG,
Widdowson PS,
Lock EB
(1996)
The role of glutathione in L-2-chloropropionic acid induced cerebellar granule cell necrosis in the rat.
Arch Toxicol
70:724-735[Medline].
-
Ye ZC,
Rothstein JD,
Sontheimer H
(1999)
Compromised glutamate transport in human glioma cells: reduction-mislocalization of sodium-dependent glutamate transporters and enhanced activity of cystine-glutamate exchange.
J Neurosci
19:10767-10777[Abstract/Free Full Text].
Copyright © 2002 Society for Neuroscience 0270-6474/02/22188028-06$05.00/0
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TOP
ABSTRACT
INTRODUCTION
